The study of metabolism has made great strides elucidating the metabolic pathways that sustain life in the cell. This investigation has produced a complex map of metabolites and enzymes that describe the chemical transformations that are possible in an organism. With the sequencing of entire genomes it is possible to foresee the conclusion of this phase of study such that the function of each gene product encoded in a genome will be described and every metabolite will be cataloged. At that point, we will have a static view of the chemistry possible in an exquisitely dynamic organism. To gain insight into the dynamic structure of metabolism, new tools must be developed to measure transient levels of metabolites in a living cell. The research proposed here will develop tools for the real-time monitoring of intra-cellular metabolites in vivo using native and engineering biosensors founded on transcriptional regulators. Although the tools developed here will be of value for the study of metabolism in general, we will focus our research on the development of biosensors for the purpose of directing the evolution of metabolism. The identification of chemical compounds has traditionally relied on expensive and cumbersome analytical instruments that require considerable expertise to operate. The data generated by these machines must be interpreted by a trained scientist to accurately determine the presence or absence of a specific compound in a complex sample. Microorganisms face a similar problem as they must constantly sample their chemical environme nt to identify the most efficient source of carbon, nitrogen and other cellular building materials. Microbes accomplish this task through a multitude of biological sensing systems (biosensors) that determine which metabolic pathway should be expressed at any given time. Exploiting these sensor-actuator systems to detect target compounds will not only provide the tools for the next phase of metabolism study, but will also deliver methodology to direct the metabolic machinery of the cell to make target compounds. These systems also may be used to detect toxic chemicals in the environment such as groundwater contamination or chemical warfare agents. We propose the development of chemical sensor/actuator systems to detect target analytes. We will demonstrate the utility of these biosensors as tools for metabolic engineering, as well as develop methodogy applicable for building any desired biosensor. To demonstrate that a biosensor can be used to direct the evolution of a metabolic pathway, we will first use a native transcriptional regulator to report on the relative concentrations of intracellular target compounds and we will use this regulator to direct the evolution of a biosynthetic pathway to make more of the target compound. Next, we will develop a selection cassette for the evolution of new transcriptional regulators with desired properties. Finally, we will use the selection cassette to evolve new transcriptional regulators to detect specific target molecules. Accordingly, the specific tasks of this proposal are (1) to use the endogenous transcriptional activator PrpR to direct the evolution of a propionate production pathway, (2) to build a reporter/selection (RS) cassette for the evolution of new biosensors, and (3) to evolve new DitR-based biosensors that sense different target compounds.